TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC 50 s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to ϳ2,000-fold for those with the D168V or D168I mutation, compared to the EC 50 for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC 50 s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC 50 s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.Hepatitis C is a blood-borne infection that can ultimately result in severe liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma (7). The chronic nature of the disease and the significant possibility of long-term liver damage have led to the current global health burden, with an estimated 180 million people being infected, of whom 130 million are chronic hepatitis C virus (HCV) carriers (54).The current standard-of-care therapy for HCV-infected patients consists of a combination of weekly injected pegylated alpha interferon (Peg-IFN-␣) and twice-daily oral ribavirin. Treatment of HCV genotype 1-infected patients with this regimen for 48 weeks has a limited success rate (a 40 to 50% sustained virological response [SVR]) and is associated with a wide range of side effects, including flu-like symptoms, anemia, and depression, leading to treatment discontinuation in a significant proportion of patients (31, 48). Therefore, specifically targeted antiviral therapies for hepatitis C (STAT-C) have been a major focus of drug discovery efforts. Treatments with several NS3/4A protease inhibitors and NS5A and NS5B polymerase inhibitors, alone or in combination with Peg-IFN-␣-ribavirin, have recently shown encouraging results in clinical trials (17,36).HCV NS3 is an essential, bifunctional, multidomain protein that possesses protease and RNA helicase activiti...

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the ␣-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three ␤-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 Å, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation.

The K103N substitution is a frequently observed HIV-1 RT mutation in patients who do not respond to combinationtherapy. The drugs Efavirenz, MSC194 and PNU142721 belong to the recent generation of NNRTIs characterized by an improved resistance profile to the most common single point mutations within HIV-1 RT, including the K103N mutation. In the present study we present structural observations from Efavirenz in complex with wild-type protein and the K103N mutant and PNU142721 and MSC194 in complex with the K103N mutant. The structures unanimously indicate that the K103N substitution induces only minor positional adjustments of the three inhibitors and the residues lining the binding pocket. Thus, compared to the corresponding wild-type structures, these inhibitors bind to the mutant in a conservative mode rather than through major rearrangements. The structures implicate that the reduced inhibitory efficacy should be attributed to the changes in the chemical environment in the vicinity of the substituted N103 residue. This is supported by changes in hydrophobic and electrostatic interactions to the inhibitors between wild-type and K103N mutant complexes. These potent inhibitors accommodate to the K103N mutation by forming new interactions to the N103 side chain. Our results are consistent with the proposal by Hsiou et al. [Hsiou, Y., Ding, J., Das, K., Clark, A.D. Jr, Boyer, P.L., Lewi, P., Janssen, P.A., Kleim, J.P., Rosner, M., Hughes, S.H. & Arnold, E. (2001) J. Mol. Biol. 309, 437-445] that inhibitors with good activity against the K103N mutant would be expected to have favorable interactions with the mutant asparagines side chain, thereby compensating for resistance caused by stabilization of the mutant enzyme due to a hydrogen-bond network involving the N103 and Y188 side chains.Keywords: drug-resistance; HIV; NNRTI; reverse transcriptase.The use of highly active antiretroviral therapy (HAART) involving multidrug combinations has significantly reduced the death rates of HIV-1 infected individuals receiving such treatment [1]. Inhibitors of the HIV-1 reverse transcriptase (RT) constitute a cornerstone in this therapy and are commonly used in combination with inhibitors of the HIV-1 protease. The RT inhibitors belong to two classes, the nucleoside inhibitors and the non-nucleoside inhibitors (NNRTI). Whereas the NRTIs are nucleoside analogues with chain-terminating properties and affinity to active site residues, the NNRTIs include a wide range of series of chemical compounds characterized by noncompetitive binding to an allosteric site some 10 Å away from the active site. Structural comparison of RT in complex with template/primer and NNRTIs together with native RT complexes have shown that the NNRTIs inhibit the polymerase activity through long-range and short-range structural distortions in several of the RT subdomains. The distortions involve repositioning of residues in the nonnucleoside binding pocket (NNIBP) that impose steric impediments on the thumb subdomain flexibility forcing it to remain in the ...

New efficient routes to enantiopure phospholipids, starting from (S)-glycidol, are described. Lysophosphatidic acids and phosphatidic acids were obtained in good overall yields from (S)-glycidol, in only three and four steps, respectively. Moreover, the strategy can also be used to produce phosphatidylcholines in three steps. Using dialkylphosphoramidites, (S)-glycidol was phosphorylated to give (R)-1-O-glycidyl dialkyl phosphates. Regiospecific epoxide opening, using hexadecanol or cesium palmitate, followed by phosphate deprotection, provided lysophosphatidic acids. 2-O-Esterification prior to phosphate deprotection provided 1,2-O-diacyl and 1-O-alkyl-2-O-acyl phosphatidic acids. Phosphorylation of (S)-glycidol using phosphorus oxychloride followed by in situ treatment with choline tosylate produced (R)-glycidyl phosphocholine. Subsequent nucleophilic opening of the epoxide using cesium palmitate produced 1-O-palmitoyl-sn-glycero-3-phosphocholine, which has been used in syntheses of phosphatidylcholines.

New and potent inhibitors of the malarial aspartic proteases plasmepsin (Plm) I and II, from the deadliest malaria parasite Plasmodium falciparum, have been synthesized utilizing Suzuki coupling reactions on previously synthesized bromobenzyloxy-substituted statine-like inhibitors. The enzyme inhibition activity has been improved up to eight times by identifying P1 substituents that effectively bind to the continuous S1-S3 crevice of Plasmepsin I and II. By replacement of the bromo atom in the P1 p-bromobenzyloxy-substituted inhibitors with different aryl substituents, several inhibitors exhibiting K(i) values in the low nanomolar range for both Plm I and II have been identified. Some of these inhibitors are also effective in attenuating parasite growth in red blood cells, with the best inhibitors, compounds 2 and 4, displaying 70% and 83% inhibition, respectively, at a concentration of 5 microM. The design was partially guided by the X-ray crystal structure disclosed herein of the previously synthesized inhibitor 1 in complex with plasmepsin II.